U.S. patent number 10,428,369 [Application Number 15/176,710] was granted by the patent office on 2019-10-01 for analyzing microdroplet outline size and adjusting channel pressure to alter microdroplet size.
This patent grant is currently assigned to Bio-Rad Laboratories, Inc.. The grantee listed for this patent is Bio-Rad Laboratories, Inc.. Invention is credited to Darren Roy Link, Benjamin J. Miller, Qun Zhong.
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United States Patent |
10,428,369 |
Miller , et al. |
October 1, 2019 |
Analyzing microdroplet outline size and adjusting channel pressure
to alter microdroplet size
Abstract
The invention generally relates to methods and systems for
manipulating droplet size. In certain aspects, the invention
provides methods for manipulating droplet size that include forming
droplets of aqueous fluid surrounded by an immiscible carrier
fluid, and manipulating droplet size during the forming step by
adjusting pressure exerted on the aqueous fluid or the carrier
fluid.
Inventors: |
Miller; Benjamin J. (Littleton,
MA), Zhong; Qun (Lexington, MA), Link; Darren Roy
(Lexington, MA) |
Applicant: |
Name |
City |
State |
Country |
Type |
Bio-Rad Laboratories, Inc. |
Hercules |
CA |
US |
|
|
Assignee: |
Bio-Rad Laboratories, Inc.
(Hercules, CA)
|
Family
ID: |
48135176 |
Appl.
No.: |
15/176,710 |
Filed: |
June 8, 2016 |
Prior Publication Data
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Document
Identifier |
Publication Date |
|
US 20160281140 A1 |
Sep 29, 2016 |
|
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
|
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14173974 |
Feb 6, 2014 |
|
|
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13554655 |
Jul 20, 2012 |
8658430 |
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61509837 |
Jul 20, 2011 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
B05B
7/0012 (20130101); B01F 13/0062 (20130101); B01L
3/502761 (20130101); B05B 1/08 (20130101); B05B
1/26 (20130101); B01F 3/0807 (20130101); B01F
15/00357 (20130101); B01L 3/502784 (20130101); B05B
1/02 (20130101); C12Q 1/6806 (20130101); B01L
2400/0487 (20130101); B01L 2300/0867 (20130101); B01L
2300/14 (20130101); B01L 7/525 (20130101); B01L
2200/0647 (20130101); Y10T 436/2575 (20150115); B01L
2200/025 (20130101); B01L 2200/143 (20130101); B01L
2200/141 (20130101); B01L 2200/148 (20130101) |
Current International
Class: |
C12Q
1/6806 (20180101); B01F 13/00 (20060101); B01F
15/00 (20060101); B05B 7/00 (20060101); B05B
1/26 (20060101); B05B 1/08 (20060101); B01F
3/08 (20060101); B01L 7/00 (20060101); B01L
3/00 (20060101); B05B 1/02 (20060101) |
Other References
Song, Helen, Delai L. Chen, and Rustem F. Ismagilov. "Reactions in
droplets in microfluidic channels." Angewandte chemie international
edition 45.44 (2006): 7336-7356. cited by examiner.
|
Primary Examiner: Vanni; G Steven
Attorney, Agent or Firm: Brown Rudnick LLP Meyers; Thomas
C.
Parent Case Text
RELATED APPLICATION
The present application is a continuation of U.S. Nonprovisional
Ser. No. 14/173,974, filed Feb. 6, 2014, which is a continuation of
U.S. Nonprovisional Ser. No. 13/554,655, filed Jul. 20, 2012, now
U.S. Pat. No. 8,658,430, which claims benefit of and priority to
U.S. Provisional No. 61/509,837, filed Jul. 20, 2011, the content
of each is incorporated by reference herein in its entirety.
Claims
What is claimed is:
1. A method of analyzing a microdroplet, comprising: forming a
microdroplet in a microfluidic channel; obtaining a projected image
of the microdroplet in the microfluidic channel, wherein the image
includes a microdroplet outline; measuring an outside diameter of
the microdroplet outline; measuring an inside diameter of the
microdroplet outline; analyzing the size of the microdroplet by
using a midpoint diameter of the microdroplet outline, wherein the
midpoint diameter is the average of the outside diameter and the
inside diameter; and adjusting pressure inside the microfluidic
channel to alter the size of the microdroplet.
2. The method of claim 1, further comprising generating a
calibration curve for the size of the microfluidic channel, and
using the calibration curve in analyzing the size of the
microdroplet.
3. The method of claim 2, wherein the microfluidic channel is
measured with a plurality of reference microdroplets of known
volumes.
4. The method of claim 1, wherein the microdroplet comprises an
aqueous fluid surrounded by an immiscible carrier fluid.
5. The method of claim 4, wherein the carrier fluid is a
fluorocarbon oil.
6. The method of claim 4, wherein the carrier fluid comprises a
first surfactant.
7. The method of claim 6, wherein the first surfactant is a fluoro
surfactant.
8. The method of claim 6, wherein the aqueous fluid comprises a
second surfactant.
9. The method of claim 1, further comprising flowing a plurality of
microdroplets in the microfluidic channel, and obtaining an image
of each microdroplet in the microfluidic channel.
10. The method of claim 9, wherein the plurality of microdroplets
travel at the same velocity.
11. The method of claim 9, wherein the plurality of microdroplets
travel at different velocities.
12. The method of claim 9, wherein the plurality of microdroplets
are of substantially similar size.
13. The method of claim 9, wherein the plurality of microdroplets
are of different sizes.
14. The method of claim 9, wherein the aqueous fluid comprises a
sample.
15. The method of claim 14, wherein the sample is a target nucleic
acid.
16. The method of claim 15, wherein the aqueous fluid further
comprises a primer and reagents for an amplification reaction.
17. The method of claim 16, wherein the amplification reaction is a
polymerase chain reaction (PCR).
18. The method of claim 1, further comprising obtaining feedback
from the projected image.
19. The method of claim 18, further comprising adjusting the size
of the droplet after obtaining the feedback.
Description
FIELD OF THE INVENTION
The invention generally relates to methods and systems for
manipulating fluidic droplet size.
BACKGROUND
The ability to precisely manipulate fluidic streams enhances the
use and effectiveness of microfluidic devices. Typically, networks
of small channels provide a flexible platform for manipulation of
small amounts of fluids. Certain microfluidic devices utilize
aqueous droplets in an immiscible earner fluid. The droplets
provide a well-defined, encapsulated microenvironment that
eliminates cross contamination and changes in concentration due to
diffusion or surface interactions.
Microfluidic devices for performing biological, chemical, and
diagnostic assays generally include at least one substrate
containing one or more etched or molded channels. The channels are
generally arranged to form individual fluid circuits, each circuit
including a sample fluid channel, an immiscible carrier fluid
channel, and an outlet channel. The channels of each circuit may be
configured such that they meet at a junction so that droplets of
aqueous fluid surrounded by carrier fluid are formed at the
junction and flow into the outlet channel. In some cases, the
outlet channel of each circuit is connected to a main channel that
receives all of the droplets from the different fluidic circuits
and flows them to an analysts module. In other cases, the outlet
channels connect to exit ports to carry the droplets to a
collection vessel.
Since each fluidic circuit may have different samples, and because
different compositions (e.g., concentration and/or length of
nucleic acid) from different samples affect how droplets form,
droplets of different sizes may be produced by each circuit. A
problem with droplets of different sizes flowing through the same
channel is that the droplets travel at different velocities.
Droplets traveling at different velocities may cause unwanted
collisions or unwanted coalescence of droplets in the channel. Thus
it is important that individual fluidic circuits produce droplets
of uniform size so that the droplets travel at the same velocity in
the channel and do nor collide or coalesce in an unwanted
manner.
Droplets are typically generated one at a time at a junction
between an aqueous fluid and an immiscible earner fluid. Droplet
volume and frequency (the number of droplet generated per unit
time) are determined by geometrical factors such as the
cross-sectional area of the channels at the junction and the
fluidic properties such as the fluid viscosities and surface
tensions as well as the infusion rates of the aqueous and carrier
fluids. To control the volume of the aqueous droplet, within a
range, droplet volume can be adjusted by tuning the oil infusion
rate through the junction. This is readily achieved with a pressure
regulator on the carrier fluid stream. In some cases it is
desirable to have multiple junctions operating as separate circuits
to generate droplets and have independent control over the oil
infusion rates through each circuit. This is readily achieved by
using separate pressure regulators for each aqueous stream and each
carrier fluid stream. A simpler and lower cost system would have a
single carrier oil source at a single pressure providing a flow of
carrier oil through each system. The problem with such a system is
that in adjusting the pressure to regulate the How of carrier oil
in one circuit the carrier oil in all circuits would be effected
and independent control over droplet volume would be compromised.
Thus, it is important to have a means whereby at a fixed carrier
oil pressure the flow of carrier oil in each of the circuits can be
independently controlled to regulate droplet volume.
SUMMARY
The invention generally relates to methods and systems for
manipulating droplet size. The invention recognizes that in a
fluidic circuit, changing the pressure exerted on the aqueous phase
changes the flow rate of the immiscible carrier fluid. Changing the
flow rate of the immiscible fluid manipulates the size of the
droplet. Thus, adjusting pressure, which changes flow rate, adjusts
droplet size. Pressure adjustments may be made independent of one
another such that the pressure exerted on the aqueous phase in
individual fluidic circuits can be adjusted to produce droplets of
uniform size from the different fluidic circuits. In this manner,
droplets produced from different fluidic circuits travel at the
same velocity in a main channel and do not collide or coalesce in
an unwanted manner.
In certain aspects, the invention provides methods for manipulating
droplet size that involve forming droplets of aqueous fluid
surrounded by an immiscible carrier fluid, and manipulating droplet
size during the forming step by adjusting pressure exerted on the
aqueous fluid or the carrier fluid. Methods of the invention
involve forming a sample droplet. Any technique known in the an for
forming sample droplets may be used with methods of the invention.
An exemplary method involves flowing a stream of sample fluid so
that the sample stream intersects two opposing streams of flowing
carrier fluid. The carrier fluid is immiscible with the sample
fluid. Intersection of the sample fluid with the two opposing
streams of flowing carrier fluid results in partitioning of the
sample fluid into individual sample droplets. The carrier fluid may
be any fluid that is immiscible with the sample fluid. An exemplary
carrier fluid is oil. In certain embodiments, the carrier fluid
includes a surfactant, such as a fluorosurfactant.
Methods of the invention may be conducted in microfluidic channels.
As such, in certain embodiments, methods of the invention may
further involve flowing the droplet channels and under microfluidic
control. Methods of the invention further involve measuring the
size of a generated droplet. Any method known in the art may be
used to measure droplet size. Preferable methods involve realtime
image analysis of the droplets, which allows for a feedback loop to
be created so that droplet size may be adjusted in real-time. In
certain embodiments, measuring the droplet site is accomplished by
taking an image of the droplet and measuring a midpoint of an
outline of the droplet image, as opposed to measuring an inside or
an outside of the droplet.
Another aspect of the invention provides methods for forming
droplets of a target volume that include flowing an aqueous fluid
through a first channel, flowing an immiscible carrier fluid
through a second channel, forming an aqueous droplet surrounded by
the carrier fluid, and adjusting resistance in the first or second
channels during the forming step to adjust volume of the droplets,
thereby forming droplets of a target volume.
Another aspect of the invention provides methods for forming
substantially uniform droplets that involve flowing a plurality of
different aqueous fluids through a plurality of different channels,
flowing an immiscible carrier fluid through a carrier fluid
channel, forming substantially uniform droplets of the different
aqueous fluids, each droplet being surrounded by the carrier fluid,
by independently adjusting resistance in the different
channels.
Another aspect of the invention provides microfluidic chips that
include a substrate, and a plurality of channels, in which at least
two of the channels include pressure regulators, the pressure
regulators being independently controllable. Generally, the
plurality of channels include at least one aqueous fluid channel,
at least one immiscible earner fluid channel, at least one outlet
channel, and a main channel. In certain embodiments, the channels
are configured to form microfluidic circuits, each circuit
including an aqueous fluid channel, a carrier fluid channel, and an
outlet channel. The channels of each circuit meet at a junction
such that droplets of aqueous fluid surrounded by carrier fluid are
formed at the junction and How into the outlet channel. Each outlet
channel of each circuit is connected to the main channel. The
channels may be etched or molded into the substrate. The channels
may be open channels or enclosed channels. Droplets may be
collected in a vessel on the device or off of the device.
Another aspect of the invention provides droplet systems that
include a microfluidic chip dial include a substrate, and a
plurality of channels, in which at least two of the channels
include pressure regulators, the pressure regulators being
independently controllable; and a pressure source coupled to the
chip.
Other aspects and advantages of the invention are provided in the
following description and claims.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a drawing showing a device for droplet formation.
FIG. 2 is a drawing showing a device for droplet formation.
FIG. 3 is a graph showing droplet size sensitivity to changes in
aqueous flow rate when using positive displacement pumping.
FIG. 4 is a graph showing droplet size sensitivity to changes in
aqueous flow rate when using pressure driven pumping.
FIG. 5 shows a diagram of a single fluidic circuit.
FIG. 6 is a drawing illustrating that the same volume drop is
subject to extreme changes in the lighting but the midpoint is
always the same. From left to right, the intensity of the lighting
decreases but the midpoint of the outline is always the same.
FIGS. 7A-C provides three graphs that demonstrate the differences
in the droplet measuring techniques, and the projected area
required to produce 5 pL drops when using the inside, outside and
midpoint of a droplet image.
FIG. 8 is a schematic illustrating measurement of droplet size
using the midpoint technique described herein.
FIG. 9 is a schematic diagram showing a microfluidic interconnect
as described in the Specification, containing a plurality of
aqueous fluid ports and an immiscible fluid port for use in methods
of the invention.
FIG. 10 is a schematic diagram showing an apparatus as described in
the Specification showing the microfluidic interconnect shown in
FIG. 9 with a manifold overlay and immiscible fluid storage.
FIG. 11 is a schematic diagram showing the relationship between the
microfluidic interconnect of FIG. 9 with a microfluidic chip for
use in methods of the invention.
DETAILED DESCRIPTION
The invention generally relates to methods and systems for
manipulating droplet size. In certain aspects, the invention
provides methods for manipulating droplet size that involve forming
droplets of aqueous fluid surrounded by an immiscible carrier
fluid, and manipulating droplet size during the forming step by
adjusting pressure exerted on the aqueous fluid or the carrier
fluid.
Droplet Formation
Methods of the invention involve forming sample droplets. In
certain embodiments, the droplets include nucleic acid from
different samples. In particular embodiments, each droplet includes
a single nucleic acid template, a single protein molecule or single
cell. The droplets are aqueous droplets that are surrounded by an
immiscible carrier fluid. Methods of forming such droplets are
shown for example in Link et al. (U.S. patent application numbers
2008/0014589, 2008/0003142, and 2010/0137163). Stone et al. (U.S.
Pat. No. 7,708,949 and U.S. patent application number
2010/0172803). Anderson et al. (U.S. Pat. No. 7,041,481 and which
reissued as RE41,780) and European publication number EP2047910 to
Raindance Technologies Inc. The content of each of which is
incorporated by reference herein in its entirety.
FIG. 1 shows an exemplary embodiment of a device 100 for droplet
formation. Device 100 includes an inlet channel 101, and outlet
channel 102, and two carrier fluid channels 103 and 104. Channels
101, 102, 103, and 104 meet at a junction 105. Inlet channel 101
flows sample fluid to the junction 105. Carrier fluid channels 103
and 104 flow a carrier fluid that is immiscible with the sample
fluid to the junction 105. Inlet channel 101 narrows at its distal
portion wherein it connects to junction 105 (See FIG. 2). Inlet
channel 101 is oriented to be perpendicular to carrier fluid
channels 103 and 104. Droplets are formed as sample fluid flows
from inlet channel 101 to junction 105, where the sample fluid
interacts with flowing carrier fluid provided to the junction 105
by carrier fluid channels 103 and 104. Outlet channel 102 receives
the droplets of sample fluid surrounded by carrier fluid.
The sample fluid is typically an aqueous buffer solution, such as
ultrapure water (e.g., 18 mega-ohm resistivity, obtained, for
example by column chromatography), 10 mM Tris HCl and 1 mM EDTA
(TE) buffer, phosphate buffer saline (PBS) or acetate buffer. Any
liquid or buffer that is physiologically compatible with enzymes
can be used. The carrier fluid is one that is immiscible with the
sample fluid. The carrier fluid can be a non-polar solvent, decane
(e.g., tetradecane or hexadecane), fluorocarbon oil, silicone oil
or another oil (for example, mineral oil).
In certain embodiments, the earner fluid contains one or more
additives, such as agents which reduce surface tensions
(surfactants). Surfactants can include Tween, Span,
fluorosurfactants, and other agents that are soluble in oil
relative to water. In some applications, performance is improved by
adding a second surfactant to the sample fluid. Surfactants can aid
in controlling or optimizing droplet size, flow and uniformity, for
example by reducing the shear force needed to extrude or inject
droplets into an intersecting channel. This can affect droplet
volume and periodicity, or the rate or frequency at which droplets
break off into an intersecting channel. Furthermore, the surfactant
can serve to stabilize aqueous emulsions in fluorinated oils from
coalescing.
In certain embodiments, the droplets may be coated with a
surfactant. Preferred surfactants that may be added to the carrier
fluid include, but are not limited to, surfactants such as
sorbitan-based carboxylic acid esters (e.g., the "Span"
surfactants, Fluka Chemika), including sorbitan monolaurate (Span
20), sorbitan monopalmitate (Span 40), sorbitan monostearate (Span
60) and sorbitan monooleate (Span 80), and perfluorinated
polyethers (e.g., DuPont Krytox 157 FSL, FSM, and/or FSH). Other
non-limiting examples of non-ionic surfactants which may be used
include polyoxyethylenated alkylphenols (for example, nonyl-,
p-dodecyl-, and dinonylphenols), polyoxyethylenated straight chain
alcohols, polyoxyethylenated polyoxypropylene glycols,
polyoxyethylenated mercaptans, long chain carboxylic acid esters
(for example, glyceryl and polyglycerl esters of natural fatty
acids, propylene glycol, sorbitol, polyoxyethylenated sorbitol
esters, polyoxyethylene glycol esters, etc.) and alkanolamines
(e.g., diethanolamine-fatty acid condensates and
isopropanolamine-fatty acid condensates).
In certain embodiments, the carrier fluid may be caused to flow
through the outlet channel so that the surfactant in the carrier
fluid coats the channel walls. In one embodiment, the
fluorosurfactant can be prepared by reacting the perflourinated
polyether DuPont Krytox 157 FSL, FSM, or FSH with aqueous ammonium
hydroxide in a volatile fluorinated solvent. The solvent and
residual water and ammonia can be removed with a rotary evaporator.
The surfactant can then be dissolved (e.g., 2.5 wt %) in a
fluorinated oil (e.g., Flourinert (3M), which then serves as the
carrier fluid.
Manipulating Droplet Size
The invention recognizes that in a fluidic circuit, changing the
pressure exerted on the aqueous phase changes the flow rate of the
immiscible carrier fluid. Changing the flow rate of the immiscible
fluid manipulates the size of the droplet. Thus, adjusting
pressure, which changes flow rate, adjusts droplet size. Pressure
adjustments may be made independently of each other such that die
pressure exerted on the aqueous phase in individual fluidic
circuits can be adjusted to produce droplets of uniform size from
the different fluidic circuits. In this manner, droplets produced
from different fluidic circuits travel at the same velocity in a
main channel and do not collide or coalesce in an unwanted manner.
When the pressure is the variable parameter used for control, there
is coupling between the aqueous and immiscible carrier fluid (e.g.,
oil) channels in an individual circuit. Therefore, any change to
the aqueous pressure has an impact on the pressure at the nozzle
and in turn affects the flow rate of the immiscible carrier fluid
(IMF). For instance, increasing P.sub.Aq, decreases Q.sub.IMF and
vice-versa. Proper design of the resistances in both the aqueous
and immiscible earner fluid channels controls the degree of
coupling that can be expected when making a change to one or more
of the input pressures. This in turn controls the sensitivity of
the change in drop volume as a function of P.sub.A.
For comparison, the sensitivity of drop size to a change in flow
rate is compared using both a positive displacement pump and a
pressure driven system. FIG. 3 is a graph showing droplet size
sensitivity to changes in aqueous flow rate when using positive
displacement pumping. FIG. 4 is a graph showing droplet size
sensitivity to changes in aqueous flow rate when using pressure
driven pumping. Oil was used as the immiscible fluid for these
comparisons. Using a similar chip with a similar circuit, a
positive displacement pump yields a 10% change in drop volume when
changing the flow rate by a factor of two. The pressure driven
system yields a 2% change in drop volume for every psi of change in
P.sub.A. If the pressure was doubled, a 60% change in drop size
could be expected when using the pressure driven system. Using a
similar circuit, pressure gives 6.times. better control over the
droplet volume when the aqueous channel is adjusted.
In certain embodiments, multiple fluidic circuits are used to
produce droplets that all flow into a main channel. Proper design
of die fluidic circuits, specifically by adjusting the fluidic
resistance in both the aqueous and oil channels, controls the
degree of influence that adjustments to the aqueous pressure has on
each of the circuits, resulting in all of the circuits producing
droplets of the same size. Changes in droplet size as a result of
changes in pressure and flow rate can be modeled using the below
calculations.
FIG. 5 shows a diagram of a single fluidic circuit for calculation
purposes. One of skill in the an will recognize that the
calculations shown herein may be applied to multiple fluidic
circuits. (A) represents an immiscible carrier fluid channel, (B)
represents an aqueous channel, (C) represents a junction of
channels (A) and (B) where aqueous phase and immiscible carrier
fluid phase meet to form droplets of the aqueous phase surrounded
by the immiscible carrier fluid, and (D) represents outlet channel
that receives the droplets. P.sub.A represents the pressure of the
immiscible carrier fluid in the immiscible carrier fluid channel.
P.sub.B represents the pressure of the aqueous fluid in the aqueous
fluid channel P.sub.C represents the pressure at the junction of
channels (A) and (B), P.sub.A, and P.sub.C are ail greater than 0,
and P.sub.D is equal to 0 because channel (D) is open to the
atmosphere. Q.sub.AC represents the flow rate of the immiscible
fluid, Q.sub.BC represents the flow rate of the aqueous fluid, and
Q.sub.CD represents the flow rate of droplets in channel (D),
R.sub.AC represents the fluid resistance in the immiscible carrier
fluid channel R.sub.BC represents the fluidic resistance in the
aqueous channel, and R.sub.CD represents the fluidic resistance in
the (D) channel. Equations and expressions for Q.sub.AC and
Q.sub.BC are as follows: PA-PC=QAC(RAC) Equation 1; PB-PC=QBC(RBC)
Equation 2; and PC=QCD(RCD)=(QAC+QBC)RCD Equation 3. Assuming that
PA, PB, RAC, RBC, and RCD are known, then the three unknowns are
PC, QAC, and QBC, OAC and QBC can be solved for as follows:
.function..times..function..function..times..times..times..times..functio-
n..times..function..function..times..times. ##EQU00001##
The sensitivities of the follow rates (Q) to changes in pressure
(P) are determined by obtaining partial derivatives of QAC and QBC
with respect to PA and PB, which yields:
.delta..times..times..delta..times..times..function..function..times..tim-
es..delta..times..times..delta..times..times..function..function..times..t-
imes..delta..times..times..delta..times..times..function..function..times.-
.times..times..times..delta..times..times..delta..times..times..function..-
function..delta..times..times..delta..times..times..times..times.
##EQU00002## Assuming that PA=PA+.delta.PA then:
'.times..times..delta..times..times..function..times..times..times..times-
..times.'.times..times..delta..times..times..function..times..times..times-
. ##EQU00003## Similarly, assuming that F''B=PB+.delta.B then:
''.times..times..delta..times..times..function..times..times..times..time-
s..times.''.times..times..delta..times..times..function..times..times..tim-
es. ##EQU00004## Substituting chip dPCR 1,3 specifies into the
above and assuming PA.apprxeq.PB, thus neglecting PA-PB containing
terms yields:
'.times..times..delta..times..times..times..times.'.times..times..delta..-
times..times..times..times.''.times..times..delta..times..times..times..ti-
mes..times..times.''.times..times..delta..times..times..times..times.
##EQU00005##
The results in FIG. 4 show that changing PA from 28 psi to 30 psi
results in Q.sub.BC going from 577 .mu.L/hr to 558 .mu.L/hr, -3.3%
change. The above model predicts a -3.1% change in QB.sub.1 which
is in agreement with the actually results data.
In certain embodiments, the system may be configured such that the
circuits produce droplets of different size to allow for controlled
droplet coalescence in the main channel. The fluidic circuits are
arranged and controlled to produce an interdigitation of droplets
of different sizes flowing through a channel. Such an arrangement
is described for example in Link et al. (U.S. patent application
numbers 2008/0014589, 2008/0003142, and 2010/0137163) and European
publication number EP2047910 to Raindance Technologies Inc. Due to
size variance, the smaller droplet will travel at a greater
velocity than the larger droplet and will ultimately collide with
and coalesce with the larger droplet to form a mixed droplet.
Another benefit of the added resistance in both channels next to
the nozzle occurs during priming. Simultaneous arrival of both the
aqueous and carrier liquids is difficult to produce reliably. If
the carrier fluid enters the aqueous channel and travels all the
way back into the filter elements, the aqueous and carrier liquids
begin to mix and emulsify before the nozzle. This mixing
interference causes significant variability in the size of the
generated droplets. The added resistance nest to the nozzle
eliminates, the mixing interference by creating a path, of
relatively high resistance without emulsifying features that are in
the filter. Therefore, if the carrier fluid arrives at the nozzle
first it will, travel both into the aqueous resistor and towards
the outlet of the chip. The outlet of the chip has a resistance
that is much smaller than the aqueous resistor and therefore the
majority of the carrier fluid will flow in that direction. This
gives the aqueous liquid time to reach the nozzle before the
carrier fluid enters the filter feature.
Droplet Measurement
The volume of an individual droplet is measured using real-time
image analysis. This in turn is fed back into a control loop where
a known projected area is targeted and equal to a given droplet
volume. Microfluidic chips are calibrated using a 3 point reference
emulsion of know volumes to generate calibration curves for each
channel. The idea is that the midpoint of the outline of a
projected droplet image is always the same regardless of the
lighting. This demonstrated in FIG. 6, which is a drawing
illustrating that the same volume drop is subject to extreme
changes in the lighting but the midpoint is always the same. From
left to right, the intensity of the lighting decreases but the
midpoint of the outline is always the same. In contrast to
determining the projected area of the inside of the drop, which is
difficult due to chip and lighting imperfections and variability,
or the outside of the drop, which is also quite sensitive to
lighting and chip imperfections, methods of the invention use the
midpoint of the outline of a projected droplet image, which is
always the same regardless of the lighting and chip imperfections.
Using the midpoint "flattens out" the imperfections and is
significantly less sensitive to outside influences on projected
drop size. FIGS. 7A-C provides three graphs that demonstrate the
differences in the droplet measuring techniques, and the projected
area required to produce 5 pL drops when using the inside, outside
and midpoint of a droplet image. Finding both the outside and
inside projected area allows you calculate the outside and inside
diameters. Calculating the average of the outside and inside
diameters gives you the midpoint diameter. From there an estimated
projected area is calculated from the midpoint diameter (See FIG.
8).
Nucleic Acid Target Molecules
One of skill in the art will recognize that methods and systems of
the invention are not limited to any particular type of sample, and
methods and systems of the invention may be used with any type of
organic, inorganic, or biological molecule. In particular
embodiments the droplets include nucleic acids. Nucleic acid
molecules include deoxyribonucleic acid (DNA) and/or ribonucleic
acid (RNA). Nucleic acid molecules can be synthetic or derived from
naturally occurring sources. In one embodiment, nucleic acid
molecules are isolated from a biological sample containing a
variety of other components, such as proteins, lipids and
nontemplate nucleic acids. Nucleic acid template molecules can be
obtained from any cellular material, obtained from an animal,
plant, bacterium, fungus, or any other cellular organism. In
certain embodiments, the nucleic acid molecules are obtained from a
single cell. Biological samples for use in the present invention
include viral particles or preparations. Nucleic acid molecules can
be obtained directly from an organism or from a biological sample
obtained from an organism, e.g., from blood, urine, cerebrospinal
fluid, seminal fluid, saliva, sputum, stool and tissue. Any tissue
or body fluid specimen may be used as a source for nucleic acid for
use in the invention. Nucleic acid molecules can also be isolated
from cultured cells, such as a primary cell culture or a cell line.
The cells or tissues from which template nucleic acids are obtained
can be infected with a virus or other intracellular pathogen. A
sample can also be total RNA extracted from a biological specimen,
a cDNA library, viral, or genomic DNA.
Generally, nucleic acid can be extracted from a biological sample
by a variety of techniques such as those described by Maniatis, et
al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor,
N.Y., pp. 280-281 (1982). Nucleic acid molecules may be
single-stranded, double-stranded, or double-stranded with
single-stranded regions (for example, stem- and
loopstructures).
Target Amplification
Methods of the invention further involve amplifying a target
nucleic acid(s) in a droplet. Amplification refers to production of
additional copies of a nucleic acid sequence and is generally
carried out using polymerase chain reaction or other technologies
well known in the art (e.g., Dieffenbach and Dveksler, PCR Primer,
a Laboratory Manual, Cold Spring Harbor Press, Plainview. N.Y.
[1995]. The amplification reaction may be any amplification
reaction known in the art that amplifies nucleic acid molecules,
such as polymerase chain reaction, nested polymerase chain
reaction, polymerase chain reaction-single strand conformation
polymorphism, ligase chain reaction (Barany F. (1991) PNAS
88:189-193; Barany F. (1991) PCR Methods and Applications 1:5-16),
ligase detection reaction (Barany F. (1991) PNAS 88:189-193),
strand displacement amplification and restriction fragments length
polymorphism, transcription based amplification system, nucleic
acid sequence-based amplification, rolling circle amplification,
and hyper-branched rolling circle amplification.
In certain embodiments, the amplification reaction is the
polymerase chain reaction. Polymerase chain reaction (PCR) refers
to methods by K. B. Mullis (U.S. Pat. Nos. 4,683,195 and 4,683,202,
hereby incorporated by reference) for increasing concentration of a
segment of a target sequence in a mixture of genomic DNA without
cloning or purification.
The process for amplifying the target sequence includes introducing
an excess of oligonucleotide primers so a DNA mixture containing a
desired target sequence, followed by a precise sequence of thermal
cycling in the presence of a DNA polymerase. The primers are
complementary to their respective strands of the double stranded
target sequence.
To effect amplification, primers are annealed to their
complementary sequence within the target molecule. Following
annealing, the primers are extended with a polymerase so as to form
a new pair of complementary strands. The steps of denaturation,
primer annealing and polymerase extension can be repeated many
times (i.e., denaturation, annealing and extension constitute one
cycle; there can be numerous cycles) to obtain a high concentration
of an amplified segment of a desired target sequence. The length of
the amplified segment of the desired target sequence is determined
by relative positions of the primers with respect to each other,
and therefore, this length is a controllable parameter.
Methods for performing PCR in droplets are shown for example in
link et al. (U.S. patent application numbers 2008/0014589,
2008/0003142, and 2010/0137163), Anderson et al. (U.S. Pat. No.
7,041,481 and which reissued as RE41,780) and European publication
number EP2047910 to Raindance Technologies Inc. The content of each
of which is incorporated by reference herein in its entirety.
The sample droplet may be pre-mixed with a primer or primers and
other reagents for on amplification reaction, or the primer or
primers and other reagents for an amplification reaction may be
added to the droplet. In some embodiments, fluidic circuits are
controlled to produce droplets of different sizes to result in
controlled merging of droplets. In those embodiments, sample
droplets are created by segmenting the starting sample and merging
that droplet with a second set of droplets including one or more
primers for the target nucleic acid in order to produce final
droplets. The merging of droplets can be accomplished using, for
example, one or more droplet merging techniques described for
example in link et al. (U.S. patent application numbers
2008/0014589, 2008/0003142, and 2010/0137163) and European
publication number EP2047910 to Raindance Technologies Inc.
In embodiments involving merging of droplets, two droplet formation
modules are used. A first droplet formation module produces the
sample droplets that on average contain a single target nucleic
acid. A second droplet formation module produces droplets that
contain reagents for a PCR reaction. Such droplets generally
include Taq polymerase, deoxynucleotides of type A, C, G and T,
magnesium chloride, and forward and reverse primers, all suspended
within an aqueous buffer. The second droplet also includes
detectably labeled probes for detection of the amplified target
nucleic acid, the details of which are discussed below. In
embodiments that start with a pre-mix of sample and reagents for a
PCR reaction, the pre-mix includes all of the above described
components.
The droplet formation modules are arranged and controlled to
produce an interdigitation of sample droplets and PCR reagent
droplets flowing through a channel. Such an arrangement is
described for example in Link et al. (U.S. patent application
numbers 2008/0014589, 2008/0003142, and 2010/0137163) and European
publication number EP2047910 to Raindance Technologies Inc.
A sample droplet is then caused to merge with a PCR reagent
droplet, producing a droplet that includes Taq polymerase,
deoxynucleotides of type A, C, G and T, magnesium chloride, forward
and reverse primers, detectably labeled probes, and the target
nucleic acid. Droplets may be merged for example by: producing
dielectrophoretic forces on the droplets using electric field
gradients and then controlling the forces to cause the droplets to
merge; producing droplets
of different sizes that thus travel at different velocities, which
causes the droplets to merge; and producing droplets having
different viscosities that thus travel at different velocities,
which causes the droplets to merge with each other. Each of those
techniques is further described in Link et al. (U.S. patent
application numbers 2008/001458, 2008/0003142, and 2010/0137163)
and European publication number EP2047910 to Raindance Technologies
Inc. Further description of producing and controlling
dielectrophoretic forces on droplets to cause the droplets to merge
is described in Link et al. (U.S. patent application number
2007/0003442) and European Patent Number EP2004316 to Raindance
Technologies Inc.
Primers can be prepared by a variety of methods including but not
limited to cloning of appropriate sequences and direct chemical
synthesis using methods well known in the art (Narang et al.,
Methods Enzymol., 68:90 (1979); Brown et al., Methods Enzymol.,
68:109 (1979)). Primers can also be obtained from commercial
sources such as Operon Technologies, Amersham Pharmacia Biotech,
Sigma, and Life Technologies. The primers can have an identical
melting temperature. The lengths of the primers can be extended or
shortened at the 5' end or the 3' end to produce primers with
desired melting temperatures. Also, the annealing position of each
primer pair can be designed such that the sequence and, length of
the primer pairs yield the desired melting temperature. The
simplest equation for determining the melting temperature of
primers smaller than base pairs is the Wallace Rule
(Td=2(A+T)+4(G+C)). Computer programs can also be used to design
primers, including but not limited to Array Designer Software
(Arrayit Inc.), Oligonucleotide Probe Sequence Design Software for
Genetic Analysis (Olympus Optical Co.), NetPrimer, and DNAs is from
Hitachi Software Engineering. The TM (melting or annealing
temperature) of each primer is calculated using software programs
such as Oligo Design, available from Invitrogen Corp.
Once final droplets have been produced, the droplets are thermal
cycled, resulting in amplification of the target nucleic acid in
each droplet. In certain embodiments, the droplets are flowed
through a channel in a serpentine path between heating and cooling
lines to amplify the nucleic acid in the droplet. The width and
depth of the channel may be adjusted to set the residence time at
each temperature, which can be controlled to anywhere between less
than a second and minutes.
In certain embodiments, the three temperature zones are used tor
the amplification reaction. The three temperature zones are
controlled to result in denaturation of double stranded nucleic
acid (high temperature zone), annealing of primers (low temperature
zones), and amplification of single stranded nucleic acid, to
produce double stranded nucleic acids (intermediate temperature
zones). The temperatures within these zones fall within ranges well
known in the art for conducting PCR reactions, See for example,
Sambrook et al. (Molecular Cloning, A Laboratory Manual, 3rd
edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
N.Y., 2001).
In certain embodiments, the three temperature zones are controlled
to have temperatures as follows: 95.degree. C. (T.sub.H),
55.degree. C. (T.sub.L), 72.degree. C. (T.sub.M). The prepared
sample droplets flow through the channel at a controlled rate. The
sample droplets first pass the initial denaturation zone (T.sub.H)
before thermal cycling. The initial preheat is an extended zone to
ensure that nucleic acids within the sample droplet have denatured
successfully before thermal cycling. The requirement for a preheat
zone and the length of denaturation time required is dependent on
the chemistry being used in the reaction. The samples pass into the
higher temperature zone, of approximately 95.degree. C., where the
sample is first separated into single stranded DNA in a process
called denaturation. The sample then flows to the low temperature,
of approximately 55.degree. C., where the hybridization process
takes place, during which the primers anneal to the complementary
sequences of the sample. Finally, as the sample flows through the
third medium temperature, of approximately 72.degree. C., the
polymerase process occurs when the primers are extended along the
single strand of DNA with a thermostable enzyme.
The nucleic acids tinder go the same thermal cycling and chemical
reaction as the droplets passes through each thermal cycle as they
flow through the channel. The total number of cycles in the device
is easily altered by an extension of thermal zones. The sample
undergoes the same thermal cycling and chemical reaction as it
passes through N amplification cycles of the complete thermal
device.
In other embodiments, the temperature zones are controlled to
achieve two individual temperature zones for a PCR reaction. In
certain embodiments, the two temperature zones are controlled to
have temperatures as follows: 95.degree. C. (T.sub.H) and
60.degree. C. (T.sub.L). The sample droplet optionally flows
through an initial preheat zone before entering thermal cycling.
The preheat zone may be important for some chemistry for activation
and also to ensure that double stranded nucleic acid in the
droplets are fully denatured before the thermal cycling reaction
begins. In an exemplary embodiment, the preheat dwell length
results in approximately 10 minutes preheat of the droplets at the
higher temperature.
The sample droplet continues into the high temperature zone, of
approximately 95.degree. C., where the sample is first separated
into single stranded DNA in a process called denaturation. The
sample then flows through the device to the low temperature zone,
of approximately 60.degree. C., where the hybridization process
takes place, during which the primers anneal to the complementary
sequences of the sample. Finally the polymerase process occurs when
the primers are extended along the single strand of DNA with a
thermostable enzyme. The sample undergoes the same thermal cycling
and chemical reaction as it passes through each thermal cycle of
the complete device. The total number of cycles in the device is
easily altered by an extension of block length and tubing.
Target Detection
After amplification, droplets are flowed to a detection module for
detection of amplification products. The droplets may be
individually analyzed and detected using any methods known in the
art, such as detecting for the presence or amount of a reporter.
Generally, the detection module is in communication with one or
more detection apparatuses. The detection apparatuses can be
optical or electrical detectors or combinations thereof. Examples
of suitable detection apparatuses include optical waveguides,
microscopes, diodes, light stimulating devices, (e.g., lasers),
photo multiplier tubes, and processors (e.g., computers and
software), and combinations thereof, which cooperate to detect a
signal representative of a characteristic, marker, or reporter, and
to determine and direct the measurement or the sorting action at a
sorting module. Further description of detection modules and
methods of detecting amplification products in droplets are shown
in link et al. (U.S. patent application numbers 2008/0014589,
2008/0003142, and 2010/0137163) and European publication number
EP2047910 to Raindance Technologies Inc.
In certain embodiments, amplified target are detected using
detectably labeled probes. In particular embodiments, the
detectably labeled probes are optically labeled probes, such as
fluorescently labeled probes. Examples of fluorescent labels
include, but are not limited to, Atto dyes,
4-acetamido-4'-isothiocyanatostilbene-2,2'disulfonic acid; acridine
and derivatives: acridine, acridine isothiocyanate;
5-(2'-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS);
4-amino-N-[3-vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonate;
N-(4-anilino-1-naphthyl)maleimide; anthranilamide; BODIPY;
Brilliant Yellow; coumarin and derivatives; coumarin,
7-amino-4-methylcoumarin (AMC, Coumarin 120),
7-amino-4-trifluoromethylcouluarin (Coumaran 151); cyanine dyes;
cyanosine; 4'6-diamimdino-2-phenylindole (DAPI);
5'5''-dibromopyrogallol-sulfonaphthalein (Bromopyrogallol Red);
7-diethylamino-3-(4'-isothiocyanatophenyl)-4-methylcumarin;
diethylenetriamine pentaacetate;
4,4'-diisothiocyanatodihydro-stilbene-2,2'-disulfonic acid;
4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid;
5-[dimethylamino]naphthalene-1-sulfonyl chloride (DNS,
dansylchloride); 4-dimethylaminophenylazophenyl-4'-isothiocyanate
(DABITC); eosin and derivatives; eosin, eosin isothiocyanate,
erythrosin and derivatives; erythrosin B, erythrosin,
isothiocyanate; ethidium; fluorescein and derivatives;
5-carboxyfluorescein (FAM),
5-(4,6-dichlorotriazin-2-yl)aminofluorescein (DTAF),
2',7'-dimethoxy-4'5'-dichloro-6-carboxyfluorescein, fluorescein,
fluorescein isothiocyanate, QFITC, (XRITC); fluorescamine; IR144;
IR1446; Malachite Green isothiocyanate; 4-methylumbelliferoneortho
cresolphthalein; nitrotyrosine; pararosaniline; Phenol Red;
B-phycoerythrin; o-phthaldialdehyde; pyrene and derivatives;
pyrene, pyrene butyrate, succinimidyl 1-pyrene; butyrate quantum
dots; Reactive Red 4 (Cibacron.TM. Brilliant Red 3B-A) rhodamine
and derivatives: 6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine
(R6G), lissamine rhodamine B sulfonyl chloride rhodamine (Rhod),
rhodamine B, rhodamine 123, rhodamine X isothiocyanate,
sulforhodamine B, sulforhodamine 101, sulfonyl chloride derivative
of sulforhodamine 101 (Texas Red);
N,N,N',N'tetramethyl-6-carboxyrhodamine (TAMRA); tetramethyl
rhodamine; tetramethyl rhodamine isothiocyanate (TRITC);
riboflavin; rosolic acid; terbium chelate derivatives; Cy3; Cy5;
Cy5.5; Cy7; IRD 700; IRD 800; La Jolta Blue; phthalo cyanine; and
naphthalo cyanine. Preferred fluorescent labels are cyanine-3 and
cyanine-5. Labels other than fluorescent labels are contemplated by
the invention, including other optically-detectable labels.
During amplification, fluorescent signal is generated in a TaqMan
assay by the enzymatic degradation of the fluorescently labeled
probe. The probe contains a dye and quencher that are maintained in
close proximity to one another by being attached to the same probe.
When in close proximity, the dye is quenched by fluorescence
resonance energy transfer to the quencher.
Certain probes are designed that hybridize to the wild-type of the
target, and other probes are designed that hybridize to a variant
of the wild-type of the target. Probes that hybridize to the
wild-type of the target have a different fluorophore attached than
probes that hybridize to a variant of the wild-type of the target.
The probes that hybridize to a variant of the wild-type of the
target are designed to specifically hybridize to a region in a ICR
product that contains or is suspected to contain a single
nucleotide polymorphism or small insertion or deletion.
During the PCR amplification, the amplicon is denatured allowing
the probe and PCR primers to hybridize. The PCR primer is extended
by Taq polymerase replicating the alternative strand. During the
replication process the Taq polymerase encounters the probe which
is also hybridized to the same strand and degrades it. This
releases the dye and quencher from the probe which are then allowed
to move away from each other. This eliminates the FRET between the
two, allowing the dye to release its fluorescence. Through each
cycle of cycling more fluorescence is released. The amount of
fluorescence released depends on the efficiency of the PCR reaction
and also the kinetics of the probe hybridization. If there is a
single mismatch between the probe and the target sequence the probe
will not hybridize as efficiently and thus a fewer number of probes
are degraded during each round of PCR and thus less fluorescent
signal is generated. This difference in fluorescence per droplet
can be detected and counted. The efficiency of hybridization can be
affected by such things as probe concentration, probe ratios
between competing probes, and the number of mismatches present in
the probe.
Droplet Sorting
Methods of the invention may further include sorting the droplets.
A sorting module may be a junction of a channel where the flow of
droplets can change direction to enter one or more other channels,
e.g., a branch channel, depending on a signal received in
connection with a droplet interrogation in the detection module.
Typically, a sorting module is monitored and/or under the control
of the detection module, and therefore a sorting module may
correspond to the detection module. The sorting region is in
communication with and is influenced by one or more sorting
apparatuses.
A sorting apparatus includes techniques or control systems, e.g.,
dielectric, electric, electro-osmotic, (micro-) valve, etc. A
control system can employ a variety of sorting techniques to change
or direct the flow of molecules, cells, small molecules or
particles into a predetermined branch channel. A branch channel is
a channel that is in communication with a sorting region and a main
channel. The main channel can communicate with two or more branch
channels at the sorting module or branch point, forming, for
example, a T-shape or a Y-shape. Other shapes and channel
geometries may be used as desired. Typically, a branch channel
receives droplets of interest as detected by the detection module
and sorted at the sorting module. A branch channel can have an
outlet module and/or terminate with a well or reservoir to allow
collection or disposal (collection module or waste module,
respectively) of the molecules, cells, small molecules or
particles. Alternatively, a branch channel may be in communication
with other channels to permit additional sorting.
A characteristic of a fluidic droplet may be sensed and/or
determined in some fashion, for example, as described herein (e.g.,
fluorescence of the fluidic droplet may be determined), and, in
response, an electric field may be applied or removed from the
fluidic droplet to direct the fluidic droplet to a particular
region (e.g. a channel). In certain embodiments, a fluidic droplet
is sorted or steered by inducing a dipole in the uncharged fluidic
droplet (which may be initially charged or uncharged), and sorting
or steering the droplet using an applied electric field. The
electric field may be an AC field, a DC field, etc. For example, a
channel containing fluidic droplets and carrier fluid, divides into
first and second channels at a branch point. Generally, the fluidic
droplet is uncharged. After the branch point, a first electrode is
positioned near the first channel, and a second electrode is
positioned near the second channel. A third electrode is positioned
near the branch point of the first and second channels. A dipole is
then induced in the fluidic droplet using a combination of the
electrodes. The combination of electrodes used determines which
channel will receive the flowing droplet. Thus, by applying the
proper electric field, the droplets can be directed to either the
first or second channel as desired. Further description of droplet
sorting is shown for example in Link et al. (U.S. patent
application numbers 2008/0014589, 2008/0003142, and 2010/0137163)
and European publication number EP2047910 to Raindance Technologies
Inc.
Release From Droplets
Methods of the invention may further involve releasing the enzymes
from the droplets for further analysis. Methods of releasing
contents from the droplets are shown for example in Link et al.
(U.S. patent application numbers 2008/0014589, 2008/0003142, and
2010/0137163) and European publication number EP2047910 to
Raindance Technologies Inc.
In certain embodiments, sample droplets are allowed to cream to the
top of the carrier fluid. By way of non-limiting example, the
carrier fluid can include a perfluorocarbon oil that can have one
or more stabilizing surfactants. The droplet rises to the top or
separates from the carrier fluid by virtue of the density of the
carrier fluid being greater than that of the aqueous phase that
makes up the droplet. For example, the perfluorocarbon oil used in
one embodiment of the methods of the invention is 1.8, compared to
the density of the aqueous phase of the droplet, which is 1.0.
The creamed liquids are then placed onto a second earner fluid
which contains a destabilizing surfactant, such as a perfluorinated
alcohol (e.g. 1H,1H,2H,2H-Perfluoro-1-octanol). The second carrier
fluid can also be a perfluorocarbon oil. Upon mixing, the aqueous
droplets begins to coalesce, and coalescence is completed by brief
centrifugation at low speed (e.g., 1 minute at 2000 rpm in a
microcentrifuge). The coalesced aqueous phase can now be removed
and the further analyzed.
Microfluidic Chips
Microfluidic chips for performing biological, chemical, and
diagnostic assays are described in U.S. Published Patent
Application No. US2008/0003142 and US2008/0014589, the content of
each of which is incorporated by reference herein in its entirety.
Such microfluidic devices generally include at least one substrate
having one or more microfluidic channels etched or molded into the
substrate, and one or mote interconnects (fluid interface). The one
or more interconnects contain inlet modules that lead directly into
the microfluidic channels, and serve to connect the microfluidic
channel to a means for introducing a sample fluid to the channel.
The one or more interconnects also serve to form a seal between the
microfluidic substrate and the means for introducing a sample. The
one or more interconnects can be molded directly into the
microfluidic substrate, as one or more individual pieces, or as a
single, monolithic self-aligning piece. The interconnect may also
be a separate piece and the entire assembly (the manifold,
microfluidic chip, and interconnect) can be modular as well. An
exemplary interconnect is shown in FIG. 9, which shows the
interconnect with immiscible fluid port 901 and aqueous fluid port
902. FIG. 10 shows the interconnect integrated with a manifold
having oil reservoir 1003 and a microfluidic chip thereunder. FIG.
11 shows the interconnect 1104 integrated with a microfluidic chip
1105 with the manifold (not shown) removed.
Microfluidic chips according to the invention include a substrate
defining at least one internal channel and at least one port in
fluid communication with the channels. In one particular
embodiment, a chip of the invention includes a top plate adhered to
a bottom plate to form the substrate with the channel(s) and
port(s). The top plate can include the port(s), and the bottom
plate can include the channel(s), such that when these two plates
are brought together and adhered to each other the combination
forms the substrate with the channel(s) and the port(s). The
microfluidic chip can be injection molded from a variety of
materials. Preferably the microfluidic chip is injection molded
using a cyclic olefin copolymer (COC) or cyclic olefin polymer
(COP) or blend of COC and COP.
Chips of the invention include one or more fluidic circuits. Each
circuit including a sample fluid channel, an immiscible carrier
fluid channel, and an outlet channel. The channels of each circuit
are configured such that they meet at a junction so that droplets
of aqueous fluid surrounded by carrier fluid are formed at the
junction an flow into the outlet channel. The outlet channel of
each circuit is connected to a main channel that receives all of
the droplets from the different fluidic circuits and flows the
droplets to different modules in the chip for analysis. In certain
embodiments, each fluid circuit carries a different aqueous sample
fluid in order to produce different sample droplets. In other
embodiments, the fluidic circuits all carry the same aqueous sample
fluid, and thus produce the same sample droplets.
A pressure source, optionally coupled to electronic pressure
regulators, is used to pump fluids through multiple microfluidic
channels in parallel. Multiple pressure regulators control the
aqueous inputs. The immiscible carrier fluid input is under gain
control for all channels simultaneously. In this configuration,
there is independent control of individual circuits to adjust
projected area to obtain a target droplet volume. Droplet volume is
measured either relatively or absolutely (depending on the
application) via real-time image analysis. Proper design of the
microfluidic circuits is required to obtain sensitive and precise
control of the droplet volume in all channels.
Pressure driven flow allows for the replacement of expensive
mechanical parts with inexpensive pneumatic control products.
Pressure driven flow is instantaneous and pulse-free. Taking
advantage of circuits in parallel, constant pressure driven flow
instantly adjusts to changes in resistance in any and all channels
without affecting any of the other channels. Any pressure sources
known in the art may be used with chips of the invention. In
certain embodiments, the pressure source is coupled to electronic
regulators. When coupled to an electronic regulator, the pressure
source may be an external compressor with a reservoir for pumping
compressed nitrogen, argon or air. In embodiments that do not used
electronic regulators, an internal air cylinder with a linear
actuator is applied.
The regulators should be of a type capable of regulating gas
pressure from about 0 to about 5 atm in 100 evenly spaced
increments (0-10 V, step=0.1 V). Each aqueous input is
independently driven and controlled by a separate pressure
regulator. The immiscible fluid lines are controlled in a gain
control fashion, where one regulator is used to drive and control
the flow of immiscible fluid through the entire system.
Incorporation by Reference
References and citations to other documents, such as patents,
patent applications, patent publications, journals, books, papers,
web contents, have been made throughout this disclosure. All such
documents are hereby incorporated herein by reference in their
entirety for all purposes.
EQUIVALENTS
The invention may be embodied in other specific forms without
departing from the spirit or essential characteristics thereof. The
foregoing embodiments are therefore to be considered in all
respects illustrative rather than limiting on the invention
described herein.
* * * * *